Displacement chromatography has been developed in the middle of the 20th century mainly for the separation of anorganic metal ions or rare earth elements, organic substances, and later also for small biomolecules like antibiotics, amino acids and peptides. At the beginning of the 80s this type of chromatography has begun to be applied for the separation of proteins. The theory and practical examples are summarized in various reviews, like Katti et al. 1992 (Katti A. M., Guichon G. A. Fundamentals in Nonlinear Chromatography: Prediction of experimental Profiles in “Advances in Chromatography” (J. C. Gidding, E. Grushka, P. R. Brown, eds.) Vol 31, pp. 1-118, 1992), Shukla et al. 2000 (Shukla A. A., Cramer S. M. Bioseparation by Displacement Chromatography in “Handbook of Bioseparations” (Ajuha S. ed.) Vol 2, pp. 379-415, Academic Press 2000) or Karlsson et al. 2000 (Karlsson E., Rydén L., Brewer J. Ion Exchange Chromatography in “Protein Purification” (Janson J-Ch., Rydén L. eds.) second edition, pp. 154-206, Wiley-Liss 1998).
In chromatography different modes of elution exists including isocratic elution, gradient elution and displacement. In displacement chromatography the column is eluted with a substance, the displacer (usually of low molecular weight) with a high affinity for the adsorbent (resin), whereas other substances (e.g. product and impurities) move at the same velocity through the column ahead of the displacer forming rectangular zones that follow each other in a train. An alternative mode of displacement chromatography is also called sample displacement chromatography (Veeraragavan K., Bernier A., Braendli E. 1991, Sample displacement mode chromatography: purification of proteins by use of a high-performance anion-exchange column, J. Chromatogr. Vol 541: 207-220)).
In all versions of chromatography, non-linear effects are common. These are seen as concentration-dependent retention times and asymmetric (e.g. tailing or fronting) peaks. Asymmetric peaks can result from a number of other causes as well, i.e. large extra-column volumes. In many applications, non-linear effects are disadvantageous as they decrease peak resolution and disturb quantitative evaluation. However, in preparative chromatography, heavy overloading is employed in order to increase material throughput, leading to prominent non-linear effects. A comprehensive text on non-linear chromatography has been published by G. Guiochon, S. Golshan Shirazi and A. M. Katti, Fundamentals of Preparative and Non-linear Chromatography, Academic Press, Inc. Boston (1994).
Alpha 1 antitrypsin (AAT) is intended to be used for patients suffering from genetically alpha 1 antitrypsin deficiency that can cause severe lung disease (known as hereditary emphysema). Currently, the only drug treatment available is a plasma derived AAT infused weekly requiring large doses to get therapeutic amounts of the protein to the lung where it is most needed. A recombinant AAT (rAAT) has been developed that is produced in bakers yeast Saccharomyces cerevisia without addition of any human or animal derived raw materials in the fermentation process, during the purification, and in the final formulation. The rAAT is intended to be delivered to the lung of the patients using a nebulizer. To avoid immunogenic reactions, in particular against residual amounts of the yeast host cell proteins, an rAAT product with high purity is required, especially in combination with high doses.
Thus, a strong need exists for a reliable method for obtaining highly purified rAAT. The present invention meets these and other needs.